The diaphragm is an essential mammalian skeletal muscle, as it is vital for respiration and serves as a barrier between the thoracic and abdominal cavities. Development of the diaphragm requires the integration of multiple tissues that derive from several embryonic sources. Defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common birth defect (1:3000 births) that results in severe morbidity and 50% mortality. Given the diaphragm's functional importance and the frequency and severity of CDH, an understanding of diaphragm development normally and during herniation is critical. Recently, using mouse genetics, we definitively established that the pleuroperitoneal folds, transient embryonic structures, and the muscle connective tissue fibroblasts derived from them critically regulate development of the diaphragm muscle (Merrell et al. 2015). Furthermore, we showed that mutations in these fibroblasts cause CDH. However, the molecular signals from the fibroblasts that regulate muscle development normally and are defective in CDH are not yet known. Based on preliminary studies, we hypothesize that connective tissue fibroblasts are an important source of secreted signals that recruit muscle progenitors into the developing diaphragm; regulate muscle morphogenesis; and are mis-regulated in CDH. In addition, our mouse studies (Merrell et al. 2015) suggest the novel hypothesis that somatic mosaic mutations in connective tissue fibroblasts are critical for the etiology of CDH ? a hypothesis that may explain the genetic complexity and phenotypic variability of CDH. We propose to use mouse genetic studies and CDH patient samples to test these hypotheses. Our research will elucidate the genetic, molecular, and cellular mechanisms regulating the development of the diaphragm and CDH and provide important insights into potential therapeutic targets to treat CDH.